Modified interleukin-1β converting enzyme with increased stability

ABSTRACT

Modified forms of human interleukin-1β converting enzyme (ICE) that display proteolytic activity and, furthermore, have increased stability compared to unmodified human ICE are disclosed. Nucleic acid molecules encoding a modified p10 subunit of ICE, and recombinant vectors and host cells incorporating such nucleic acid molecules, are also disclosed. A modified ICE protein of the invention can be used to cleave proteolytically ICE substrates and to identify modulators of ICE activity in screening assays. Moreover, due to its enhanced stability, the modified ICE of the invention is particularly suitable for use in the preparation of ICE crystals for X-ray crystallography.

This application is a divisional application of Ser. No. 09/248,179filed on Feb. 9, 1999, now U.S. Pat. No. 6,242,240 Issuing, which inturn is a continuation application of Ser. No. 08/573,890 filed on Dec.18, 1995, now U.S. Pat. No. 5,869,315, Issued. The contents of all ofthe aforementioned application(s) are hereby incorporated by reference.

BACKGROUND OF THE INVENTION

Interleukin-1 is a cytokine having a broad spectrum of biologicalactivities (for reviews, see e.g., Dinarello, C. A. and Wolff, S. M.(1993) New Engl. J Med. 328:106-113; and Dinarello, C. A. (1993) Trendsin Pharmacol. Sci. 4:155-159). IL-1 consists of two structurally relatedpolypeptides, interleukin-1α (IL-1α) and interleukin-1β (IL-1β). The twoforms of IL-1 are encoded by different genes and have only 27-33% aminoacid identity but they interact with the same receptor and have similarbiological activities. Included among the biological functionsattributed to IL-1 are induction of fever, sleep, anorexia andhypotension. IL-1 is also involved in the pathophysiology ofinflammatory and autoimmune diseases, including rheumatoid arthritis,septic shock, inflammatory bowel disease and insulin dependent diabetesmellitus. IL-1α has been specifically implicated in the pathophysiologyof psoriasis. IL-1 is also thought to play a role in immune responses toinfectious agents and in the pathogenesis of myeloid leukemias.

IL-1α and IL-1β are both synthesized as approximately 31 kDa precursormolecules that are subsequently processed to a mature form ofapproximately 17 kDa. IL-1α and IL-1β differ in that the precursor formof IL-1α (preIL-1α) is biologically active and most of the mature IL-1α(matIL-1α) remains cell-associated, whereas the precursor form of IL-1β(preIL-1β) must be cleaved to its mature form to become active and themature form of IL-1β (matIL-1β) is secreted from the cell. Only certaincell types process preIL-1β and secrete matIL-1β. Monocytes andmacrophages are the most efficient producers and secretors ofIL-1β,which is the most abundant form of IL-1 produced upon activation ofthese cell types.

Interleukin-1β converting enzyme (ICE) is a cytoplasmic cysteineprotease required for generating the bioactive form of theinterleukin-1β cytokine from its inactive precursor (Black, R. A. et al.(1988) J Biol. Chem. 263:9437-9442; Kostura, M. J. et al. (1989) Proc.Natl. Acad. Sci. USA 86:5227-5231; Thornberry et al. (1992) Nature356:768-774; Ceretti, D. P. et al. (1992) Science 256:97-100). ICE is amember of a family of cysteine proteases with shared homology. Othermembers of this family have been implicated in apoptosis, such as ced-3(Yuan, J. et al. (1993) Cell 75:641-652), Nedd2 (Kumar, S. et al. (1992)Biochem. Biophys. Res. Commun. 185:1155-1161; Kumar, S. et al. (1994)Genes Dev. 8:1613-1626), CPP32 (Fernandes-Alnemri, T. et al. (1994) J.Biol. Chem. 269:30761-30764), Ich-1 (Wang, L. et al. (1994) Cell78:739-750) and Ich-2 (Kamens, J. et al. (1995) J. Biol Chem.270:15250-15256; Faucheu, C. et al. (1995) EMBO J. 14:1914-1922). Thenecessity for ICE in the generation of bioactive IL-1β was demonstratedin mice in which the ICE gene had been functionally disrupted (Li, P. etal. (1995) Cell 80:401-411. Kuida, K. et al. (1995) Science26:2000-2003). Although these animals are overtly normal, they have amajor defect in the production of mature IL-1-β after stimulation withlipopolysaccharide.

In vitro studies have demonstrated that ICE cleaves prointerleukin-1β atAsp₁₁₆-Ala₁₁₇ to release the fully active 17 kDa form (Black, supra;Kostura, supra). ICE also cleaves prointerleukin-1β at Asp₂₇-Ala₂₈ torelease a 28 kDa form. Cleavage at these sites is dependent upon thepresence of aspartic acid in the P1 position (Kostura, supra, Howard, A.et al. (1991) J. Immunol. 147:2964-2969; Griffin, P. R. et al. (1991)Int. J. Mass. Spectrom. Ion. Phys. 111:131-149). However, an asparticacid in the P1 position is not sufficient for ICE specificity. Forexample, several other proteins containing Asp-X bonds, includingprointerleukin-1α, are not cleaved by ICE (Howard, supra).

ICE itself undergoes maturational processing, possibly performed in vivoby ICE itself (Thornberry, N. A. et al. (1992) Nature 3:768-774). MatureICE is generated from a 404 amino acid precursor protein by proteolyticremoval of two fragments, the N-terminal 119 amino acid “pro-domain” andthe internal residues 298-316 (Thomberry, supra). Active ICE istherefore composed of two subunits, a 20 kDa subunit (p20) encompassingresidues 120 to 297 and a 10 kDa subunit (p10) encompassing residues 317to 404. The crystal structure of ICE indicates that ICE forms atetrameric structure consisting of two p20 and two p10 subunits (Walker,N. P. C. et al. (1994), Cell 78:343-352; Wilson, K. P. et al. (1994)Nature 370:270-275). The catalytic amino acid residues of ICE areCys-285 and His-237. The side chains of four amino acid residues(Arg-179, Gln-283, Arg-341 and Ser-347) form the P1 carboxylate bindingpocket (Walker, supra; Wilson, supra).

Because of the apparently harmful role of IL-1 in many diseaseconditions, therapeutic strategies aimed at reducing the production oraction of IL-1 have been proposed. For example, one approach by which toinhibit matIL-1α production and secretion would be to block the activityof ICE with a specific ICE inhibitor. The ability to produce active ICEin vitro is therefore highly desirable to allow for the study of itsstructure and function. However, in vitro production of ICE can behampered by the instability of the protein, in particular as a result ofautocatalytic degradation which leads to inactive protein.

SUMMARY OF THE INVENTION

This invention provides modified human ICE proteins that retain theproteolytic activity of unmodified human ICE and that exhibit increasedstability in vitro compared to modified ICE. One aspect of the inventionpertains to a modified form of ICE comprising an amino acid sequencewherein an amino acid corresponding to aspartic acid at position 381 ofunmodified ICE (SEQ ID NO: 2) is replaced with a mutant amino acidstructure. This mutant amino acid structure is one that is capable offorming a salt bridge with an amino acid corresponding to arginine atposition 383 of unmodified ICE, such that the modified ICE exhibitsproteolytic activity and has increased stability compared to unmodifiedICE. The mutant amino acid structure that replaces the amino acidcorresponding to Asp381 can be a natural amino acid or a non-naturalamino acid. In the most preferred embodiment, the mutant amino acidstructure is a glutamic acid residue. In other embodiments, the mutantamino acid structure is selected from the group consisting of serine,threonine, asparagine and glutamine.

Another aspect of the invention pertains to nucleic acid moleculesencoding a modified p10 subunit of ICE. The modified p10 subunit encodedby the nucleic acid molecule comprises an amino acid sequence wherein anamino acid corresponding to aspartic acid at position 381 of unmodifiedICE (SEQ ID NO: 2) is replaced with a mutant amino acid structure. Themutant amino acid structure is one that is capable of forming a saltbridge with an amino acid corresponding to arginine at position 383 ofunmodified ICE, such that the modified p10 subunit associates with a p20subunit to form a modified ICE that retains proteolytic activity andexhibits increased stability compared to unmodified ICE. In the mostpreferred embodiment, the p10 subunit encoded by the nucleic acidmolecule has a glutamic acid residue at the position corresponding toposition 381 of unmodified ICE. In other embodiments, the p10 subunithas a serine, threonine, asparagine or glutamine residue at the positioncorresponding to position 381 of unmodified ICE.

A nucleic acid molecule of the invention encoding a modified p10 subunitof ICE can be incorporated into a recombinant expression vector. In oneembodiment, the recombinant expression vector encodes the modified p10subunit of ICE (i. e., about amino acids 317 to 404). In anotherembodiment, the recombinant expression vector encodes the modified p10subunit of ICE and also encodes the p20 subunit of ICE (i.e., aboutamino acids 120-197). For example, in one embodiment, the recombinantexpression vector encodes the p30 form of ICE, comprising about aminoacids 120-404. This p30 form of ICE undergoes maturational processing toproduce a modified p10 subunit and a p20 subunit.

The recombinant expression vectors of the invention can be introducedinto host cells to produce modified ICE proteins. In one embodiment, amodified p10 subunit is expressed recombinantly in a host cell.denatured and refolded with a p20 subunit of ICE to form a modified ICEprotein of the invention. In another embodiment, a modified p10 subunitis coexpressed with a p20 subunit in the same host cell (using eithertwo separate expression vectors or one expression vector encoding boththe p10 and p20 subunits, such as a vector encoding p30), therebyproducing a modified ICE protein of the invention.

The modified ICE proteins of the invention are cysteine proteases thatexhibit proteolytic activity against ICE substrates. Accordingly, amodified ICE protein of the invention can be used to cleave an ICEsubstrate by contacting the ICE substrate with the modified ICE suchthat the ICE substrate is cleaved. Moreover, the modified ICE proteinsof the invention can be used in screening assays to identify modulators(e.g., inhibitors or stimulators) of ICE protease activity. Stillfurther, the enhanced stability of the modified ICE proteins of theinvention makes them particularly well-suited for the preparation ofcrystalline ICE for use in X-ray cystallographic analysis (e.g., forstructure-based design of ICE inhibitors).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a photograph of an SDS-polyacrylamide gel comparing E.coli-expressed wild-type N-His ICE protein and E. coli-expressedD381E-modified N-His ICE protein.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides modified forms of human interleukin-1βconverting enzyme (ICE) which retain the proteolytic activity ofunmodified ICE yet also exhibit increased stability compared tounmodified human ICE. The invention is based, at least in part, on thediscovery that mutation of ICE at an aspartic acid at position 381(Asp-381) to glutamic acid (Glu-381) greatly increases the stability ofthe modified form of ICE (due to greatly reduced autocatalyticdegradation of the protein), while maintaining the proteolytic activityof the protein. In contrast, it was further discovered that mutation ofICE at Asp-381 to alanine (Ala-381) results in a modified ICE proteinthat, although stable, exhibits greatly reduced proteolytic activity.These results were then applied to an analysis of the crystal structureof human ICE. The crystal structure of ICE demonstrated that Asp-381forms a salt bridge with an arginine at position 383 (Arg-383). SinceGlu-381 maintains this salt bridge and Ala-381 does not, preferredmodifications in the modified ICE proteins of the invention aremodifications at a position corresponding to Asp-381 that retain thecapacity to form a salt bridge with an amino acid corresponding toArg-383.

Various aspects of the invention are described in further detail in thefollowing subsections.

I. Modified ICE Proteins

One aspect of the invention pertains to modified ICE proteins. As usedherein, the term “unmodified ICE” refers to ICE proteins having theamino acid sequence of naturally-occurring human ICE. The term“unmodified ICE” includes the precursor form of ICE having the aminoacid sequence shown in SEQ ID NO: 2 (the correspondingnaturally-occurring nucleotide sequence that encodes unmodified humanICE is shown in SEQ ID NO: 1), as well as the processed mature form ofICE composed of p20 subunits (amino acids residues 120 to 297) and p10subunits (amino acid residues 317 to 404). As used herein, the terms“aspartic acid at position 381 of unmodified ICE” (or simply Asp-381)and “arginine at position 383 of unmodified ICE” (or simply Arg-383)refer to amino acid residues within the p10 subunit using the numberingbased on the full-length precursor form of ICE as shown in SEQ ID NO: 2.

As used herein, the term “modified ICE” refers to forms of ICE thatdiffer structurally from unmodified ICE. In particular, the modified ICEproteins of the invention comprise an amino acid sequence wherein anamino acid corresponding to aspartic acid at position 381 of unmodifiedICE (as shown in SEQ ID NO: 2) is replaced with a mutant amino acidstructure, the mutant amino acid structure being capable of forming asalt bridge with an amino acid corresponding to arginine at position 383of unmodified ICE. The mutant amino acid structure is selected such thatthe modified ICE retains proteolytic activity and exhibits increasedstability compared to unmodified ICE. The term “mutant amino acidstructure” is intended to include natural amino acids and non-naturalamino acids. Non-natural amino acids include amino acid derivatives,analogues and mimetics. As used herein, a “derivative” of an amino acidrefers to a form of the amino acid in which one or more reactive groupson the compound have been derivatized with a substituent group. As usedherein an “analogue” of an amino acid refers to a compound that retainschemical structures of the amino acid necessary for functional activityof the amino acid (e.g., formation of a salt bridge with Arg-383 in ICE)yet also contains certain chemical structures that differ from the aminoacid. As used herein, a “mimetic” of an amino acid refers to a compoundin that mimics the chemical conformation of the amino acid.

Within the modified ICE proteins of the invention, an amino acidcorresponding to aspartic acid at position 381 of unmodified ICE isreplaced with a mutant amino acid structure that is capable of forming asalt bridge with an amino acid corresponding to arginine at position 383of unmodified ICE. Mutant amino acid structures that are capable offorming a salt bridge with an amino acid corresponding to arginine atposition 383 of unmodified ICE can be determined by computer modelingusing the crystal structure of unmodified ICE (determination of thecrystal structure of ICE is described in Walker, N. P. C. et al. (1994),Cell 78:343-352; and Wilson, K. P. et al. (1994) Nature 370:270-275).According to the crystal structure of unmodified ICE, Asp-381 forms asalt bridge with Arg-383. It has now been discovered that substitutionof Asp-381 with glutamic acid, which retains the ability to form a saltbridge with Arg-383, results in a modified ICE that is bothproteolytically active and more stable than unmodified ICE. In contrast,substitution of Asp-381 with an alanine. which cannot form a salt bridgewith Arg-383, results in a modified ICE that, although stable, has lostproteolytic activity, thereby demonstrating the necessity formaintenance of this salt bridge for effective proteolytic activity.

In a modified ICE of the invention, Asp-381 of unmodified ICE ispreferably substituted with another natural amino acid capable offorming a salt bridge with an amino acid corresponding to Arg-383. Inthe most preferred embodiment, Asp-381 is substituted with glutamicacid. In other embodiments, Asp-381 is substituted with an amino acidselected from the group consisting of serine, threonine, asparagine andglutamine. Other amino acids that can form a salt bridge with Arg-383include derivatives of aspartic acid and glutarnic acid, such asβ-methylaspartic acid, γ-methylglutamic acid and β-methylglutamic acid,which may occur rarely in nature or which may be non-naturalderivatives. Additional non-natural amino acids that can be used includeanalogues or mimetics of aspartic acid and glutamic acid thatincorporate modifications into the peptide backbone of the ICE protein,such as an N-methyl aspartic acid or N-methyl glutamic acid. Moreover, amodified ICE protein of the invention can be modified at the amidelinkage between Asp-381 and Gly-382, for example, the amide linkage canbe substituted with an alkyl chain (thereby inhibiting proteolyticdigestion at this linkage).

The modified ICE proteins of the invention exhibit increased stabilitycompared to unmodified ICE. Preferably, the modified ICE protein is atleast 10% more stable than unmodified ICE. More preferably, the modifiedICE protein is at least 25% more stable than unmodified ICE. Even morepreferably, the modified ICE is at least 50% more stable than unmodifiedICE. Still more preferably, the modified ICE protein is at least 75%more stable than unmodified ICE. The stability of modified andunmodified ICE proteins can be examined and quantified by direct orindirect means. For example, the degree of degradation of modified orunmodified ICE can be assessed directly and used as a measure of thestability of the protein. The degree of degradation of an ICEpreparation can be visualized directly by examining the protein bystandard sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE). Samples containing a known amount of ICE protein (modifiedvs. unmodified) are electrophoresed on a standard SDS-PAGE gel and theprotein bands are visualized by standard methods (e.g., Coomassie bluestaining, silver staining and the like). Degradation of the ICEpreparation is evidenced by the appearance of lower molecular weightbreakdown products of the p10 subunit (i. e., protein band less than 10kDa in weight, such as a predominant 7 kDa band), as described furtherin Example 2, part C and illustrated in FIG. 1. This degradation can bequantified by quantitating the relative amounts of protein in the intactp10 band as compared to the lower molecular weight bands in the ICEpreparation. The relative amount of intact p10 in a modified ICEpreparation is then compared to the relative amount of intact p10 in anunmodified ICE preparation as a measure of the stability of the modifiedICE. For example, a modified ICE preparation in which the p10 band is50% less degraded than in the unmodified ICE preparation represents amodified ICE protein that is 50% more stable than an unmodified ICEprotein.

Alternatively, stability of the modified ICE proteins of the inventioncan be assessed indirectly, for example by measuring the proteolyticactivity of modified vs. unmodified ICE preparations over time. A morestable ICE preparation exhibits greater proteolytic activity over timethan a less stable ICE preparation (see Example 2, parts A and B).Proteolytic activity can be assessed in a standard in vitro proteolysisassay (such as described in Thornberry et al. (1992) Nature 356:768-774and Example 2) using an appropriate ICE substrate. Examples ofappropriate ICE substrates include the chromogenic para-nitroanilide(pNA)-labeled peptide substrates, Acetyl-Tyr-Val-Ala-Asp-pNA(Ac-YVAD-pNA) and Acetyl-Asp-Glu-Val-Asp-pNA (Ac-DEVD-pNA), and thefluorogenic amino-4-methylcoumarin (AMC)-labeled peptide substratesAcetyl-Tyr-Val-Ala-Asp-AMC,(Ac-YVAD-AMC) and Acetyl-Asp-Glu-Val-Asp-pNA(Ac-DEVD-AMC). Ac-YVAD-pNA is described further in Reiter, L. A. (1994)Int. J. Peptide Protein Res. 43:87-96. Ac-YVAD-pNA and Ac-YVAD-AMC arecommercially available from Bachem Bioscience, Inc., King of Prussia,Pa. The rate constant of degradation of the modified ICE protein can bedetermined to quantitate the stability of the modified ICE protein.

Another aspect of the invention pertains to fusion proteins of themodified forms of ICE of the invention. The invention provides modifiedICE proteins that are fusion proteins. As used herein the term “fusionprotein” refers to a modified form of ICE in which non-ICE amino acidresidues are fused at either the amino-terminus or carboxy-terminus. Apreferred modified ICE fusion protein is one that has a polyhistidinetag (e.g., six histidine residues) at its amino terminus. Thispolyhistidine fusion moiety allows for purification of the modified ICEprotein on a nickel chelating column (Porath, J. (1992) ProteinExpression and Purification 2:263-281). Other fusion moieties can beused to facilitate protein purification, such asglutathione-S-transferase and maltose-binding protein. Preferably, amodified ICE fusion protein of the invention is prepared by recombinantDNA technology, as described further below.

II. Nucleic Acid Molecules Encoding Modified ICE Proteins

Another aspect of the invention pertains to nucleic acid moleculesencoding the modified ICE proteins of the invention. The amino acidposition that is modified in the ICE protein of the invention (i.e.,position 381 of unmodified ICE), occurs within the p10 subunit of matureICE. Accordingly, the invention provides a nucleic acid moleculeencoding a modified p10 subunit of human ICE. This modified p10 subunitencoded by the nucleic acid comprises an amino acid sequence wherein anamino acid corresponding to aspartic acid at position 381 of unmodifiedICE (SEQ ID NO: 2) is replaced with a mutant amino acid, the mutantamino acid being capable of forming a salt bridge with an amino acidcorresponding to arginine at position 383 of unmodified ICE, such thatthe modified p10 subunit associates with a p20 subunit to form amodified ICE that retains proteolytic activity and exhibits increasedstability compared to unmodified ICE. Most preferably, the nucleic acidmolecule encodes a p10 subunit in which Asp-381 is replaced withglutamic acid. In other embodiments, the nucleic acid molecule encodes ap10 subunit in which Asp-381 is replaced with an amino acid selectedfrom the group consisting of serine, threonine, asparagine andglutamine.

As used herein, the term “nucleic acid molecule” is intended to includeDNA molecules and RNA molecules. The nucleic acid molecule may besingle-stranded or double-stranded, but preferably is double-strandedDNA.

The modified ICE proteins and nucleic acid molecules of the inventionare preferably prepared using recombinant DNA technology. For example,to prepare a DNA fragment encoding a modified p10 subunit of ICE, firsta DNA fragment encoding the region of unmodified ICE encompassing aminoacid residues 317 to 404 (p10) is prepared, for example, by PCRamplification using appropriate primers designed using the nucleotidesequence of ICE shown in SEQ ID NO: 1. The oligonucleotide primers shownin SEQ ID NOs: 3 and 6 are suitable for amplifying a DNA fragmentencoding an unmodified p10 subunit. The primer of SEQ ID NO: 3corresponds to nucleotide sequences encoding the p10 amino terminus(starting at amino acid 317) and contains an EcoRI restriction site. Theprimer of SEQ ID NO: 6 is complementary to nucleotide sequences encodingthe p10 carboxy terminus (amino acids 400-404) followed by a stop codonand includes an SpeI restriction site. The DNA fragment encoding theunmodified ICE p10 subunit can then be used as a template formutagenesis to create a DNA fragment encoding a modified ICE p10subunit. Mutagenesis can be accomplished by PCR mutagenesis (asdescribed further in Example 1), or other standard methods known in theart such as site directed mutagenesis.

The nucleic acid molecules of the invention encoding modified ICE p10subunits can be used to prepare modified ICE proteins of the invention.The nucleic acid molecules can be incorporated into recombinantexpression vectors that allow for expression of the modified p10 subunitencoded therein. The modified p10 subunit can be expressed using an invitro transcription/translation system or, more preferably, is expressedby introducing the recombinant expression vector into a suitable hostcell in which the p10 subunit is then expressed (e.g., E. coli). Arecombinant expression vector of the invention can encode only themodified p10 ICE subunit (i.e., about amino acids 317-404 of ICE).Alternatively, the recombinant expression can encode both the modifiedp10 subunit and a p20 subunit (e.g., a wild-type p20 subunit, comprisingabout amino acids 120-297 of ICE)). For example, in one embodiment of arecombinant expression vector encoding both the p10 and p20 subunits,the vector carries a DNA fragment encoding amino acids 120 to 404 of ICE(referred to as p30), wherein the amino acid corresponding to Asp-381within the p10 subunit has been modified. The p30 fragment of ICE lacksthe “prodomain” from amino acids 1-119 but still contains the internalresidues 298-316, which are cleaved from the precursor form of ICE togenerate the p20 and p10 subunits. This p30 fragment is appropriatelyprocessed to p20 and p10 when expressed in a suitable host cell (e.g.,E. coli). In yet another embodiment of a recombinant expression vectorencoding both the p10 and p20 subunits, the vector encodes the entireprecurser form of ICE (i.e., amino acids 1-404), wherein the p10 subunithas been modified as described herein.

Accordingly, in one embodiment, a mature, active modified ICE protein ofthe invention is prepared by coexpression of p20 and p10 in a host cell(described further in Example 2, part B). The p20 and p10 subunits canbe coexpressed in a host cell using a p30-encoding expression vector or,alternatively, by expression of separate p20 and p10 genes, eithercarried on the same vector or carried on two separate expressionvectors. In another embodiment, a mature, active modified ICE protein ofthe invention is prepared by separately expressing the p20 and p10subunits in different host cell, recovering the two subunits, denaturingthem and renaturing the two subunits together to form mature, active ICEcomprised of associated p20 and p10 subunits (described further inExample 2, part A).

III. Recombinant Expression Vectors

Another aspect of the invention pertains to vectors, preferablyexpression vectors, containing a nucleic acid encoding a modified ICEp10 subunit of the invention, alone or together with a p20 subunit. Asused herein, the term “vector” refers to a nucleic acid molecule capableof transporting another nucleic acid to which it has been linked. Onetype of vector is a “plasmid”, which refers to a circular doublestranded DNA loop into which additional DNA segments may be ligated.Another type of vector is a viral vector, wherein additional DNAsegments may be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors are capable ofdirecting the expression of genes to which they are operatively linked.Such vectors are are referred to herein as “recombinant expressionvectors” or simply “expression vectors”. In general, expression vectorsof utility in recombinant DNA techniques are often in the form ofplasmids. In the present specification, “plasmid” and “vector” may beused interchangeably as the plasmid is the most commonly used form ofvector. However, the invention is intended to include such other formsof expression vectors, such as viral vectors, which serve equivalentfunctions.

In the recombinant expression vectors of the invention, ICE-encodingsequences are operatively linked to one or more regulatory sequences,selected on the basis of the host cells to be used for expression. Theterm “operably linked” is intended to mean that the ICE-encodingsequences are linked to the regulatory sequence(s) in a manner thatallows for expression of ICE protein (e.g., in an in vitrotranscription/translation system or in a host cell when the vector isintroduced into the host cell). The term “regulatory sequence” isintended to includes promoters, enhancers and other expression controlelements (e.g., polyadenylation signals). Such regulatory sequences aredescribed, for example, in Goeddel; Gene Expression Technology: Methodsin Enzymology 185, Academic Press, San Diego, Calif. (1990). Regulatorysequences include those that direct constitutive expression of anucleotide sequence in many types of host cell and those that directexpression of the nucleotide sequence only in certain host cells (e.g.,tissue-specific regulatory sequences). It will be appreciated by thoseskilled in the art that the design of the expression vector may dependon such factors as the choice of the host cell to be transformed, thelevel of expression of protein desired, etc. The expression vectors ofthe invention can be introduced into host cells thereby to produceproteins or peptides, including fusion proteins or peptides, encoded bynucleic acids as described herein.

The recombinant expression vectors of the invention can be designed forexpression of modified ICE proteins in prokaryotic or eukaryotic cells.For example, modified ICE proteins can be expressed in bacterial cellssuch as E. coli, insect cells (using baculovirus expression vectors)yeast cells or mammalian cells. Suitable host cells are discussedfurther in Goeddel, Gene Expression Technology: Methods in Enzymology185, Academic Press, San Diego, Calif. (1990). Alternatively, therecombinant expression vector may be transcribed and translated invitro, for example using T7 promoter regulatory sequences and T7polymerase.

Expression of proteins in prokaryotes is most often carried out in E.coli with vectors containing constitutive or inducible promotorsdirecting the expression of either fusion or non-fusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Such fusionvectors typically serve three purposes: 1) to increase expression ofrecombinant protein; 2) to increase the solubility of the recombinantprotein; and 3) to aid in the purification of the recombinant protein byacting as a ligand in affinity purification. Often, in fusion expressionvectors, a proteolytic cleavage site is introduced at the junction ofthe fusion moiety and the recombinant protein to enable separation ofthe recombinant protein from the fusion moiety subsequent topurification of the fusion protein. Such enzymes, and their cognaterecognition sequences, include Factor Xa, thrombin and enterokinase.Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc;Smith, D. B. and Johnson, K. S. (1988) Gene 67:31-40), pMAL (New EnglandBiolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) whichfuse glutathione S-transferase (GST), maltose E binding protein, orprotein A, respectively, to the target recombinant protein. In apreferred embodiment, a DNA fragment encoding ICE p30 (amino acidresidues 120-404) is cloned into an expression vector (e.g., an E. coliexpression vector) that fuses a polyhistidine sequence (e.g., sixhistidine residues) to the N-terminus of the p30-coding sequence. Thepolyhistidine fusion moiety allows for purification of the mature ICEprotein on a nickel chelating column (Porath, J. (1992) ProteinExpression and Purification 2:263-281). Polyhistidine fusion expressionvectors are commercially available (e.g., Novagen, Madison, Wis.).

Examples of suitable inducible non-fusion E. coli expression vectorsinclude pTrc (Amann et al., (1988) Gene 69:301-315) and pET 11d (Studieret al., Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990) 60-89). Target gene expression from thepTrc vector relies on host RNA polymerase transcription from a hybridtrp-lac fusion promoter. Target gene expression from the pET 11d vectorrelies on transcription from a T7 gn10-lac fusion promoter mediated by acoexpressed viral RNA polymerase (T7 gn1). This viral polymerase issupplied by host strains BL21(DE3) or HMS174(DE3) from a resident λprophage harboring a T7 gn1 gene under the transcriptional control ofthe lacUV 5 promoter.

One strategy to maximize recombinant protein expression in E. coli is toexpress the protein in a host bacteria that is impaired in its capacityto cleave proteolytically the recombinant protein (Gottesman, S., GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990) 119-128). Another strategy is to alter the nucleicacid sequence of the nucleic acid to be inserted into an expressionvector so that the individual codons for each amino acid are thosepreferentially utilized in E. coli (Sharp and Li (1986) Nucl. AcidsRes., 14:7737-7749; Wada et al., (1992) Nucl. Acids Res. 20:2111-2118).Such alteration of nucleic acid sequences of the invention can becarried out by standard DNA synthesis techniques.

In another embodiment, the ICE expression vector is a yeast expressionvector. Examples of vectors for expression in yeast S. cerivisae includepYepSec1 (Baldari et al., (1987) EMBO J. 6:229-234), pMFa (Kurjan andHerskowitz, (1982) Cell 3:933-943), pJRY88 (Schultz et al., (1987) Gene54:113-123), and pYES2 (Invitrogen Corporation, San Diego, Calif.).

Alternatively, ICE can be expressed in insect cells using baculovirusexpression vectors. Baculovirus vectors available for expression ofproteins in cultured insect cells (e.g., Sf9 cells) include the pAcseries (Smith et al., (1983) Mol. Cell. Biol. 3:2156-2165) and the pVLseries (Lucklow, V. A., and Summers, M. D., (1989) Virology 170:31-39).

In yet another embodiment, a nucleic acid of the invention is expressedin mammalian cells using a mammalian expression vector. Examples ofmammalian expression vectors include pCDM8 (Seed, B., (1987) Nature2:840) and pMT2PC (Kaufman et al. (1987), EMBO J. 6:187-195). When usedin mammalian cells, the expression vector's control functions are oftenprovided by viral regulatory elements. For example, commonly usedpromoters are derived from polyoma, Adenovirus 2, cytomegalovirus andSimian Virus 40.

IV. Recombinant Host Cells

To prepare modified ICE proteins of the invention, typically one or morerecombinant expression vectors are introduced into a suitable host cellin which the ICE protein is then expressed. The terms “host cell” and“recombinant host cell” are used interchangeably herein. It isunderstood that such terms refer not only to the particular subject cellbut to the progeny or potential progeny of such a cell. Because certainmodifications may occur in succeeding generations due to either mutationor environmental influences, such progeny may not, in fact, be identicalto the parent cell, but are still included within the scope of the termas used herein. A host cell may be any prokaryotic or eukaryotic cell.For example, modified ICE proteins may be expressed in bacterial cellssuch as E. coli, insect cells, yeast or mammalian cells. Preferably,modified ICE proteins are expressed in E. coli as described in theExamples.

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection” are intended to refer to avariety of art-recognized techniques for introducing foreign nucleicacid (e.g., DNA) into a host cell, including calcium phosphate orcalcium chloride co-precipitation, DEAE-dextran-mediated transfection,lipofection, or electroporation. Suitable methods for transforming ortransfecting host cells can be found in Sambrook et al. (MolecularCloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratorypress (1989)), and other laboratory manuals.

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest.Preferred selectable markers include those that confer resistance todrugs, such as G418, hygromycin and methotrexate. Nucleic acid encodinga selectable marker may be introduced into a host cell on the samevector as that encoding ICE protein or may be introduced on a separatevector. Cells stably transfected with the introduced nucleic acid can beidentified by drug selection (e.g., cells that have incorporated theselectable marker gene will survive, while the other cells die).

V. Uses of Modified ICE Proteins

The modified ICE proteins of the invention retain the cysteine proteaseactivity of unmodified ICE. Accordingly, the modified ICE proteins areuseful as cysteine proteases. Moreover, since a modified ICE protein ofthe invention has increased stability compared to unmodified ICE, aparticular amount of this modified ICE protein exhibits greaterproteolytic activity over time than an equal amount of unmodified ICE.Another aspect of the invention, therefore, pertains to methods forcleaving ICE substrates. The method involves contacting the substratewith a modified ICE protein of the invention such that the ICE substrateis cleaved. The term “ICE substrate” is intended to include any peptideor protein which is cleavable by ICE. ICE substrates are characterizedby an aspartic acid residue in the P1 position. Examples of ICEsubstrates include prointerleukin-1β, the chromogenic pNA-labeledpeptide substrates Ac-YVAD-pNA and Ac-DEVD-pNA and the fluorogenicAMC-labeled peptide substrates Ac-YVAD-AMC and Ac-DEVD-AMC.

The modified ICE proteins of the invention can also be used in screeningassays to identify modulators of ICE activity. The invention provides amethod for identifying a modulator of ICE protease activity, comprising:

preparing a modified ICE protein of the invention;

contacting the modified ICE with an ICE substrate in the presence of atest compound under proteolytic conditions;

measuring proteolysis of the ICE substrate in the presence of the testcompound.

comparing proteolysis of the ICE substrate in the presence of the testcompound to proteolysis of the ICE substrate in the absence of the testcompound; and

identifying the test compound as a modulator of ICE protease activity.In one embodiment, the modified ICE protein comprises an amino acidsequence wherein an amino acid corresponding to aspartic acid atposition 381 of unmodified ICE (SEQ ID NO: 2) is replaced with a mutantamino acid structure, the mutant amino acid structure being capable offorming a salt bridge with an amino acid corresponding to arginine atposition 383 of unmodified ICE, such that the modified ICE retainsenzymatic activity and exhibits increased stability compared tounmodified ICE. In the most preferred embodiment, the mutant amino acidstructure is glutamic acid. In other embodiments, the mutant amino acidstructure is selected from the group consisting of serine, threonine,asparagine and glutamine.

In one embodiment of the screening method, an inhibitor of ICE proteaseactivity is identified. In this case, the amount of proteolysis of theICE substrate in the presence of the test compound is less than theamount of proteolysis of the ICE substrate in the absence of the testcompound. In another embodiment of the screening method, an activator ofICE protease activity is identified. In this case, the amount ofproteolysis of the ICE substrate in the presence of the test compound isgreater than the amount of proteolysis of the ICE substrate in theabsence of the test compound.

Suitable ICE substrates for use in the screening assays are describedabove. Preferably, a chromogenic or fluorogenic ICE substrate is usedwhose cleavage can be detected spectrophotometrically. Alternatively,cleavage of other peptide substrate can be detected chromatographically(e.g., by HPLC). Additionally, whole proteins can be used as substrates,such as prointerleukin-1β. Whole proteins can be labelled (e.g., with³⁵S-methionine) and their cleavage products can be directly detected(e.g., by SDS-PAGE and autoradiography). Alternatively, cleavage ofwhole proteins can be detected indirectly (e.g., using an antibody thatbinds a specific cleavage product).

In addition to the foregoing uses, the modified ICE proteins of theinvention, because of their enhanced stability, are particularlywell-suited for preparing crystalline ICE for X-ray crystallographicanalysis. For example, unmodified ICE labeled at its N-terminus with apolyhistidine tag and expressed in E. coli was found to be too unstable(i.e., underwent too much autodegradation) to prepare crystals thatcould be used for X-ray crystallography. In contrast, D381E-modified ICElabeled at its N-terminus with a polyhistidine tag and expressed in E.coli was sufficiently stable for the preparation of crystals for X-raycrystallography. Accordingly, modified ICE proteins of the invention canbe expressed recombinantly as described herein (e.g., in E. coli), therecombinant ICE protein can be purified and crystals can be preparedtherefrom for use in X-ray crystallography. General methods forpreparing crystalline ICE, and performing X-ray crystallographicanalysis thereon, are described in Walker, N. P. C. et al. (1994) Cell78:343-352 and Wilson, K. P. et al. (1994) Nature 370:270-275.Furthermore, a modified ICE protein of the invention can be used inX-ray crystallography in combination with a cysteine protease inhibitor,which serves to further stabilize the protein during crystallization, asdescribed in U.S. patent application Ser. No. 08/573,896, entitled“Cysteine Protease Inhibitors and Uses Therefor”, filed Dec. 18, 1995and expressly incorporated herein by reference.

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references,patents and published patent applications cited throughout thisapplication are hereby incorporated by reference.

EXAMPLE 1

Construction of a Modified ICE

A modified form of human ICE having an aspartic acid to glutamic acidsubstitution at amino acid position 381 (D381E) was constructed bypolymerase chain reaction (PCR) mutagenesis of a DNA fragment encodingamino acids 317-404 of ICE (i.e., the p10 subunit) followed by in vivorecombination in E. coli.

PCR Amplification

To create a DNA fragment encoding p10 (amino acids 317-404) having aD381E mutation, two different DNA fragments were amplified by PCR thattogether represent the ICE 317-404 gene with a 28 base pair overlappingsequence (containing the mutation) where the two can be linked byhomologous recombination in E. coli. Additionally, the DNA fragmentswere amplified such that they had restriction sites on each end forcloning into an expression vector.

For the first fragment (referred to as Fragment A), a plasmid encodingwild-type ICE 317-404 having a methionine start codon inserted beforethe ICE alanine-317 was used as the template. This open reading framehad its codons modified to reflect the preferred codon usage of highlyexpressed genes in E. coli, according to the codon usage informationdisclosed in Sharp and Li (1986) Nucl. Acids Res., 14:7737-7749. The 5′primer (#694) had the following sequence: 5′-GGG GAA TTC ATG GCT ATC AAAAAA GCT CAC ATC GAA AAA GAC TTC ATC GCT TTC TGC-3′ (SEQ ID NO: 3). This5′ primer is complementary to the ICE 317-404 amino terminus andcontains an EcoRI restriction site. The 3′ primer (#1537) had thefollowing nucleotide sequence: 5′-TTC TGG CTG CTC AAA TGA AAA ACG AACCTT GCG GAA AAT TTC-3′ (SEQ ID NO: 4). This 3′ primer containsnucleotide sequences encoding the amino acid region ICE 368-381 andcontains 2 mutations. The first is an A to T point mutation at the 5′end (position 1) of the oligonucleotide that changes aspartic acid-381to glutamic acid. The second is a silent point mutation (T to A atposition 22 of the oligo) that eliminates a TaqI site to be used forclone screening but does not further change the amino acid sequence ofthe encoded p10 subunit. PCR amplification using these two primers andthe wildtype ICE 317-404 containing plasmid as template resulted in afragment containing an open reading frame encoding ICE 317-381 havingthe D381E mutation, with an EcoRI site for cloning at the 5′ end.

For the second fragment (referred to as Fragment B), the template wasthe same as the first. The PCR reaction utilized a 5′ primer (#1533)having the nucleotide sequence: 5′-GGT TCG TTT TTC ATT TGA GCA GCC AGAAGG TAG AGC GCA GAT G-3′ (SEQ ID NO: 5). This 5′ primer containsnucleotide sequences encoding the amino acid region ICE 373-386 andcontains the complements to the 2 mutations described in the previousparagraph. The PCR reaction utilized a 3′ primer (#518) having thefollowing sequence: 5′-CCC CAC TAG TCC TCT ATT AAT GTC CTG GGA AGA GG-3′(SEQ ID NO: 6). The 3′ primer contains nucleotide sequences encoding theamino acid region ICE 400-404 followed by a stop codon and includes anSpeI restriction site. PCR amplification using these primers and theplasmid encoding wild-type ICE 317-404 as template resulted in afragment containing an open reading frame encoding ICE 373-404,including the D381E mutation, followed at the 3′ end by the Spelrestriction site for cloning into the expression vector.

Standard PCR conditions were used to obtain the PCR fragments describedabove using 30 cycles of 94° C. denaturing step for 30 seconds,annealing for 30 seconds at 52° C., and 2 minutes elongation at 72° C.These 30 cycles were followed by 5 minutes at 73° C. for finalextension. The reactions were then held at 4° C. until ready for furtheruse. Five microliters of each of the appropriate primers at 20 μMconcentrations were used in each reaction with the following additionalreaction components: 1 μl of template DNA at 1 μg/ml, 10 μl of standard10×PCR buffer containing MgCl₂ (PE Express, Norwalk, Conn.), 8 μl ofdNTP mix containing 2.5 mM each of deoxynucleotide triphosphate (PEExpress, Norwalk, Conn.), 0.5 μl Taq polymerase (PE Express, Norwalk,Conn.) and 70 μl of deionized dH₂O for a final volume of 100 μl.

10 μl each of the two PCR fragments, Fragments A and B, from the two PCRreactions described above, were then separately run out on 1% agarosegels (FMC Bioproducts. Rockland, Me.) containing 0.5 μg/ml ethidiumbromide to visualize the resultant bands under UV Light. Both PCRproducts were observed, Fragment A at 206 base pairs and Fragment B at114 base pairs. The remaining PCR mix was extracted with an equal volumeof phenol:chloroform:isoamyl alcohol (25:24:1 v/v). The aqueous phasewas brought to 1M LiCl₂ and the DNA was precipitated by the addition of3 volumes of 100% ethanol. The DNA was collected by centrifugation. Thepellet was washed with 70% ethanol, recentrifuged and dried undervacuum. The ends of the fragments were digested with EcoRI (fragment A)or SpeI (fragment B) in 50 μl reactions containing appropriate reactionbuffer and 20 units of restriction enzyme. The reactions were allowed toproceed at 37° C. overnight.

Following, restriction enzyme digestion, Fragments A and B were thenseparately electrophoresed on 4% Metaphor agarose gels (FMC Bioproducts,Rockland, Me.) containing 0.5 μg/ml ethidium bromide to visualize theresultant bands under UV light. The expected size bands were observedand purified by electrophoresis onto DEAE paper for 10 minutes at 100 Vand eluted with buffer containing 20% ethanol, 1 M LiCl₂, 10 mM tris pH7.5, and 1 mM EDTA. The eluate was then precipitated with isopropanol,pelleted, washed with 70% ethanol, dried by speed vacuum and resuspendedin 10 μl 10 mM tris, 1 mM EDTA pH 8.0.

Ligation of Fragments A and B to Expression Vector

The two PCR fragments (A and B) were ligated to the ends of thelinearized expression vector pJAM-4. pJAM-4 is a derivative ofpBluescript II KS(+) (Stratagene, La Jolla, Calif.) consisting of thebacteriophage Lambda left promoter (pL) followed by a ribosome bindingsite which is in turn followed by a polylinker sequence starting with anEcoRI site. The vector also carries an ampicillin resistance gene, theColE1 origin and the f1 (+) origin.

The expression vector pJAM-4 (10 μg) was digested with EcoRI and SpeI bystandard methods. The linearized vector was purified from a 1% agarosegel containing 0.5 μg/ml ethidium bromide using DEAE paper, as describedabove. The linearized vector was resuspended to an estimatedconcentration of 500 ng/μl in dH₂O.

A ligation reaction was prepared containing the following reactioncomponents: 5 μl each of fragments A and B, 1 μl of linearized pJAM-4, 2μl 10×Ligase buffer (New England BioLabs, Beverly, Mass.), 1 μl T4 DNALigase (400 units) (New England BioLabs, Beverly, Mass.) and 6 μl dH₂O.Ligation was carried out overnight at 16° C. Following the ligationreaction, the ligation mix was extracted with phenol:chloroform:isoamylalcohol (25:24:1 v/v) and precipitated as described above. The resultingDNA pellet was dissolved in a final volume of 10 μl dH₂O.

Following the precipitation, the ligation products were transformed intocompetent E. coli strain MV1190 (Δ(lac-proAB), thi, supE, Δ(srl-recA)306:Tn10(Tet^(r)) [F′:traD36, proAB, lacI^(q) lacZΔM15]) (Bio-RadLaboratories, Hercules, Calif.) carrying plasmid pSE103. pSE103 is akanamycin resistant pSC101 derivative carrying the temperature sensitivebacteriophage Lambda repressor, cI⁸⁵⁷. Competent MV1190 pSE103 cellswere prepared as follows: A 3 ml seed culture in 2×YT media (Sambrook,J. et al. (1989) Molecular Cloning: A Laboratory Manual, Cold SpringHarbor, N.Y.:Cold Spring Harbor Press) was grown overnight at 30° C.from a frozen seed culture in the presence of 50 μg/ml kanamycin. Theovernight culture was used to inoculate a larger culture (100-250 ml of2×YT containing 50 μg/ml kanamycin) using a {fraction (1/50)} dilutionof the seed culture. The cells were grown with shaking at 30° C. untilthe OD₆₀₀ value reached 0.5. Cells were harvested by low speedcentrifugation, the cell pellet was resuspended in ½ original volume ofice cold 10 mM MgCl₂, 30 mM CaCl₂, 10% glycerol and the suspensionallowed to sit on ice for 30 minutes. The cells were recentrifuged atlow speed and the cell pellet was resuspended in {fraction (1/10)} theoriginal culture volume of the same solution above. The competent cellswere dispensed into 100 μl aliquots and stored at −80° C. until use.

The cells were transformed using the linear DNA that resulted from theligation of fragments A and B to the ends of the vector DNA, as follows.In a pre-chilled tube, 5 μl of ligation mix was added to 100 μl ofcompetent MV1190 pSE103 cells and incubated on ice for 30 minutes. Themixture was heat shocked at 37° C. for 2 minutes, then added to 1 ml2×YT medium and incubated for 1 hour at 30° C. 100 μl of the medium wasplated onto Luria-Bertani (LB) plates (ampicillin, 100 μg/ml; kanamycin,50 μg/ml) (Sambrook, et al., supra) and incubated at 30° C. overnight.The linear DNA, once inside the cell, undergoes a recA-independentrecombination event by virtue of the 28 base pair overlapping sequencesof Fragment A and Fragment B. This results in the recovery of an intactgene inserted into the expression vector which encodes p10 (amino acids317-404) having the D381E mutation.

Identification of Positive Clones

Transformants were screened by restriction enzyme digestion of plasmidDNA minipreps. The enzymes used were EcoRI and HindIII. Transformantscontaining inserts of the correct size were used to prepare largeramounts of pure plasmid for further analysis. The presence of the D381Emutation was demonstrated by detecting the linked mutation whichresulted in the destruction of a TaqI site (by TaqI digests) and bycomplete nucleotide sequence determination of the insert.

Expression of ICE 317-404 D381E

Expression of the mutant p10 subunit (ICE 317-404 D381E) was carried outin the protease-deficient E. coli strain CAG 597 (F-;rpoH165(am)zhg:Tn10 lacZ(am) trp(am) pho(am) supCts mal(am) rpsL) (New EnglandBioLabs, Beverly, Mass.) containing pACYC177cI⁸⁵⁷ (Kanamycin^(R)). Thep10 expression plasmid pJAM-4 ICE 317-404 D381E was transformed into thebacterial cells as described above. Transformants were selected onLuria-Bertani (LB) plates (ampicillin, 100 μg/ml; kanamycin, 50 μg/ml)(Sambrook, et al., supra) and incubated at 30° C. overnight. A singlecolony was used to inoculate a 3 ml 2×YT (ampicillin, 100 μg/ml;kanamycin, 50 μg/ml ) culture and incubated at 30° C. overnight. Oneliter of 2×YT (ampicillin, 100 μg/ml; kanamycin, 50 μg/ml ) wasinoculated with 0.5 ml of overnight seed culture and incubatedapproximately 4 hours at 30° C. with shaking. Expression of the mutantp10 was induced by shifting the incubator temperature to 42° C. and theinduced culture was incubated an additional 4 hours to allow for proteinproduction. The cells were harvested by centrifugation and the pelletsstored at −80° C. until ready for use.

EXAMPLE 2

Relative Activity and Stability of D381E-Modified ICE Compared toUnmodified ICE

The D381 E-modified form of ICE was prepared in two ways and itsactivity and/or stability was compared to wild-type (WT) ICE (i.e.,unmodified ICE). First, unmodified and D381 E-modified ICE were preparedby refolding separately-expressed p20 (WT) and p10 (WT or D381E mutant)subunits thereby to produce active, mature ICE (p20/p10). Second,unmodified and D381 E-modified ICE were prepared by expression inbacteria of a single protein (p30) that encompasses both p20 and p10,and that autoproteolytically processes to the active, p20/p10 form(either fully WT or containing a p10 subunit carrying the D381Emutation). The p30-encoding constructs also contained an amino terminalpoly-Histidine tag to allow for rapid purification of the protein, andare referred to as WT N-His ICE (for wild-type) and D381E N-His ICE (forthe D381E mutant).

A. Comparative Activity of WT and D381E-Modified ICE Refolded From p20and p10 Subunits

ICE (WT or modified) was prepared by refolding p20 and p10, which hadbeen separately expressed in E. coli, by a protocol described in U.S.patent application Ser. No. 08/242,663, entitled “ICE and ICE-LikeCompositions and Methods of Making Same”, which is expresslyincorporated herein by reference. The p20 subunit was prepared by HPLCpurification of material expressed as inclusion bodies in E. coli. Thep10 subunit (either wild-type, the D381 A-modified form or theD381E-modified form) was used only as crude inclusion bodies. A controlof HPLC-purified wild-type p10 was also included to estimate the effecton the system of the crude inclusion bodies, but the conclusions of theexperiment were derived from the comparison of the three crudematerials.

During the final step of the refolding protocol, which consists ofdialysis from a tris buffer into a HEPES buffer, ICE proteolyticactivity against an ICE substrate develops gradually over a few hours.ICE activity was monitored as a function of time in the HEPES dialysis.Four samples, each differing in the p10 species used, were compared:HPLC-pure wild-type, crude wild-type, crude D381A, and crude D381E. Theresults of this experiment are shown below in Table I, wherein ICEactivity is expressed in arbitrary units.

Table I Time into HEPES dialysis Pure WT Crude WT Crude D381A CrudeD381E   0 hours  879  95  28  370   2 hours 2165 1200 237 3246 2.5 hours4609 1332 220 3316 3.5 hours 4789 1082 252 3976   4 hours 5228 1276 n.d.3832 4.5 hours 4988 n.d. n.d. 3524   5 hours 5112 1356 584 4030 n.d. =not done

These results demonstrate that the D381 E mutation produces a modifiedform of ICE with a final yield and/or specific activity that is equal toor greater than that of unmodified ICE. In contrast, the D381A mutationproduces a modified form of ICE with substantially lower catalyticactivity than that of unmodified ICE.

B. Comparative Activity of Wild-Type and D381 E-Modified N-His ICE

ICE (WT or modified) with an N-terminal polyhistidine tag was expressedin E. coli and purified using methodology described in Kamens et al.(1995) J. Biol. Chem., 270:15250-15256. Briefly, a DNA fragment encodingamino acid residues 120 to 404 of wild-type or D381E-modified ICE (p30)was cloned into an expression vector such that the p30-encoding fragmentwas fused in-frame at its N-terminus to DNA encoding six histidineresidues. Following expression, the p30 fragment is correctly processedto p20 and p10 subunits, which associate to form mature, activeN-His-tagged ICE proteins. The N-His ICE fusion proteins then werepurified on a nickel chelating column (Porath, J. (1992) ProteinExpression and Purification 2:263-281).

The proteolytic activity of nickel-column-purified WT and D381E-modified ICE was examined using two pNA-labeled substrates,Ac-YVAD-pNA and Ac-DEVD-pNA. To perform the proteolysis assay, modifiedor unmodified N-His ICE was preincubated for 60 minutes at 30° C. in 80μl of a reaction buffer containing 100 mM HEPES, 20% (v/v) glycerol, 5mM DTT, 0.5 mM EDTA, at pH 7.5. The peptide substrate was added in 20 μlof reaction buffer containing 2.5 mM substrate and 5% DMSO solvent,giving final concentrations in the assay mixtures of 500 μM substrateand 1% DMSO. The incubation of N-His ICE with the peptide substrate at30° C. was continued, and the catalytic hydrolysis of peptide substratewas monitored by the change in absorbance of the samples at 405 nm dueto release of pNA as a function of time. Assays were performed induplicate. Samples were read using a microtiter plate reader (MolecularDevices, Sunnyvale, Calif.).

The Michaelis-Menten Km and Vmax values for the unmodified and modifiedN-His ICE using the two peptide substrates were determined by standardmethods. The results are shown below:

K_(m) values (μM) D381E WT Ac-YVAD-pNA 43 26 Ac-DEVD-pNA 44 25 V_(max)values (M ⁻¹ s⁻¹) D381E WT Ac-YVAD-pNA 0.19 0.04 Ac-DEVD-pNA 0.08 0.02

The similarities in the Km values for both substrates between the twoenzymes demonstrate that WT and D381 E-modified ICE are functionallysimilar. That the Vmax values are substantially greater with the D381Eform for both substrates, as compared to the WT form, most likelyreflects the fact that a large fraction of the WT enzyme is degraded andtherefore inactive.

C. Visualization/Quantitation of Increased Stability of D381E N-His ICE

To confirm that D381 E-modified N-His ICE was more stable (i.e.,underwent less autocatalytic degradation) than WT N-His ICE, assuggested in part B above, nickel column purified material (WT or D381E) was analyzed by standard SDS-polyacrylamide electrophoresis.Representative results are shown in FIG. 1. The WT N-His ICE preparationconsisted of the expected p20 and p10 bands as well as smaller molecularweight breakdown products, the most visible being a p7 band in FIG. 1.By sequencing analysis, this p7 band has been identified as the largercleavage product that results from cleavage of p10 at Asp-381/Gly-382.Although not visible in FIG. 1, a p3 band, corresponding to the smallercleavage product that results from cleavage of p10 at Asp-281/Gly-382,has also been identified in the WT N-His ICE preparation. In contrast tothe WT preparation, these lower molecular weight breakdown products arenot detectable in the D381E N-His ICE preparation shown in FIG. 1,whereas the p20 and p10 subunits are readily detectable. The degree ofdegradation of p10 in the WT material is estimated to be at least 50%compared to the modified material. This high degree of degradation ofthe WT sample rendered it unsuitable for use in crystallography. Incontrast, the D381E-modified material, which had no discernibledegradation of p10, was used successfully to form crystals that diffractto high resolution for X-ray crystallographic analysis of the ICEstructure.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

6 1216 base pairs nucleic acid double linear cDNA CDS 1..1212 1 ATG GCCGAC AAG GTC CTG AAG GAG AAG AGA AAG CTG TTT ATC CGT TCC 48 Met Ala AspLys Val Leu Lys Glu Lys Arg Lys Leu Phe Ile Arg Ser 1 5 10 15 ATG GGCGAA GGT ACA ATA AAT GGC TTA CTG GAT GAA TTA TTA CAG ACA 96 Met Gly GluGly Thr Ile Asn Gly Leu Leu Asp Glu Leu Leu Gln Thr 20 25 30 AGG GTG CTGAAC AAG GAA GAG ATG GAG AAA GTA AAA CGT GAA AAT GCT 144 Arg Val Leu AsnLys Glu Glu Met Glu Lys Val Lys Arg Glu Asn Ala 35 40 45 ACA GTT ATG GATAAG ACC CGA GCT TTG ATT GAC TCC GTT ATT CCG AAA 192 Thr Val Met Asp LysThr Arg Ala Leu Ile Asp Ser Val Ile Pro Lys 50 55 60 GGG GCA CAG GCA TGCCAA ATT TGC ATC ACA TAC ATT TGT GAA GAA GAC 240 Gly Ala Gln Ala Cys GlnIle Cys Ile Thr Tyr Ile Cys Glu Glu Asp 65 70 75 80 AGT TAC CTG GCA GGGACG CTG GGA CTC TCA GCA GAT CAA ACA TCT GGA 288 Ser Tyr Leu Ala Gly ThrLeu Gly Leu Ser Ala Asp Gln Thr Ser Gly 85 90 95 AAT TAC CTT AAT ATG CAAGAC TCT CAA GGA GTA CTT TCT TCC TTT CCA 336 Asn Tyr Leu Asn Met Gln AspSer Gln Gly Val Leu Ser Ser Phe Pro 100 105 110 GCT CCT CAG GCA GTG CAGGAC AAC CCA GCT ATG CCC ACA TCC TCA GGC 384 Ala Pro Gln Ala Val Gln AspAsn Pro Ala Met Pro Thr Ser Ser Gly 115 120 125 TCA GAA GGG AAT GTC AAGCTT TGC TCC CTA GAA GAA GCT CAA AGG ATA 432 Ser Glu Gly Asn Val Lys LeuCys Ser Leu Glu Glu Ala Gln Arg Ile 130 135 140 TGG AAA CAA AAG TCG GCAGAG ATT TAT CCA ATA ATG GAC AAG TCA AGC 480 Trp Lys Gln Lys Ser Ala GluIle Tyr Pro Ile Met Asp Lys Ser Ser 145 150 155 160 CGC ACA CGT CTT GCTCTC ATT ATC TGC AAT GAA GAA TTT GAC AGT ATT 528 Arg Thr Arg Leu Ala LeuIle Ile Cys Asn Glu Glu Phe Asp Ser Ile 165 170 175 CCT AGA AGA ACT GGAGCT GAG GTT GAC ATC ACA GGC ATG ACA ATG CTG 576 Pro Arg Arg Thr Gly AlaGlu Val Asp Ile Thr Gly Met Thr Met Leu 180 185 190 CTA CAA AAT CTG GGGTAC AGC GTA GAT GTG AAA AAA AAT CTC ACT GCT 624 Leu Gln Asn Leu Gly TyrSer Val Asp Val Lys Lys Asn Leu Thr Ala 195 200 205 TCG GAC ATG ACT ACAGAG CTG GAG GCA TTT GCA CAC CGC CCA GAG CAC 672 Ser Asp Met Thr Thr GluLeu Glu Ala Phe Ala His Arg Pro Glu His 210 215 220 AAG ACC TCT GAC AGCACG TTC CTG GTG TTC ATG TCT CAT GGT ATT CGG 720 Lys Thr Ser Asp Ser ThrPhe Leu Val Phe Met Ser His Gly Ile Arg 225 230 235 240 GAA GGC ATT TGTGGG AAG AAA CAC TCT GAG CAA GTC CCA GAT ATA CTA 768 Glu Gly Ile Cys GlyLys Lys His Ser Glu Gln Val Pro Asp Ile Leu 245 250 255 CAA CTC AAT GCAATC TTT AAC ATG TTG AAT ACC AAG AAC TGC CCA AGT 816 Gln Leu Asn Ala IlePhe Asn Met Leu Asn Thr Lys Asn Cys Pro Ser 260 265 270 TTG AAG GAC AAACCG AAG GTG ATC ATC ATC CAG GCC TGC CGT GGT GAC 864 Leu Lys Asp Lys ProLys Val Ile Ile Ile Gln Ala Cys Arg Gly Asp 275 280 285 AGC CCT GGT GTGGTG TGG TTT AAA GAT TCA GTA GGA GTT TCT GGA AAC 912 Ser Pro Gly Val ValTrp Phe Lys Asp Ser Val Gly Val Ser Gly Asn 290 295 300 CTA TCT TTA CCAACT ACA GAA GAG TTT GAG GAT GAT GCT ATT AAG AAA 960 Leu Ser Leu Pro ThrThr Glu Glu Phe Glu Asp Asp Ala Ile Lys Lys 305 310 315 320 GCC CAC ATAGAG AAG GAT TTT ATC GCT TTC TGC TCT TCC ACA CCA GAT 1008 Ala His Ile GluLys Asp Phe Ile Ala Phe Cys Ser Ser Thr Pro Asp 325 330 335 AAT GTT TCTTGG AGA CAT CCC ACA ATG GGC TCT GTT TTT ATT GGA AGA 1056 Asn Val Ser TrpArg His Pro Thr Met Gly Ser Val Phe Ile Gly Arg 340 345 350 CTC ATT GAACAT ATG CAA GAA TAT GCC TGT TCC TGT GAT GTG GAG GAA 1104 Leu Ile Glu HisMet Gln Glu Tyr Ala Cys Ser Cys Asp Val Glu Glu 355 360 365 ATT TTC CGCAAG GTT CGA TTT TCA TTT GAG CAG CCA GAT GGT AGA GCG 1152 Ile Phe Arg LysVal Arg Phe Ser Phe Glu Gln Pro Asp Gly Arg Ala 370 375 380 CAG ATG CCCACC ACT GAA AGA GTG ACT TTG ACA AGA TGT TTC TAC CTC 1200 Gln Met Pro ThrThr Glu Arg Val Thr Leu Thr Arg Cys Phe Tyr Leu 385 390 395 400 TTC CCAGGA CAT TAAA 1216 Phe Pro Gly His 404 amino acids amino acid linearprotein 2 Met Ala Asp Lys Val Leu Lys Glu Lys Arg Lys Leu Phe Ile ArgSer 1 5 10 15 Met Gly Glu Gly Thr Ile Asn Gly Leu Leu Asp Glu Leu LeuGln Thr 20 25 30 Arg Val Leu Asn Lys Glu Glu Met Glu Lys Val Lys Arg GluAsn Ala 35 40 45 Thr Val Met Asp Lys Thr Arg Ala Leu Ile Asp Ser Val IlePro Lys 50 55 60 Gly Ala Gln Ala Cys Gln Ile Cys Ile Thr Tyr Ile Cys GluGlu Asp 65 70 75 80 Ser Tyr Leu Ala Gly Thr Leu Gly Leu Ser Ala Asp GlnThr Ser Gly 85 90 95 Asn Tyr Leu Asn Met Gln Asp Ser Gln Gly Val Leu SerSer Phe Pro 100 105 110 Ala Pro Gln Ala Val Gln Asp Asn Pro Ala Met ProThr Ser Ser Gly 115 120 125 Ser Glu Gly Asn Val Lys Leu Cys Ser Leu GluGlu Ala Gln Arg Ile 130 135 140 Trp Lys Gln Lys Ser Ala Glu Ile Tyr ProIle Met Asp Lys Ser Ser 145 150 155 160 Arg Thr Arg Leu Ala Leu Ile IleCys Asn Glu Glu Phe Asp Ser Ile 165 170 175 Pro Arg Arg Thr Gly Ala GluVal Asp Ile Thr Gly Met Thr Met Leu 180 185 190 Leu Gln Asn Leu Gly TyrSer Val Asp Val Lys Lys Asn Leu Thr Ala 195 200 205 Ser Asp Met Thr ThrGlu Leu Glu Ala Phe Ala His Arg Pro Glu His 210 215 220 Lys Thr Ser AspSer Thr Phe Leu Val Phe Met Ser His Gly Ile Arg 225 230 235 240 Glu GlyIle Cys Gly Lys Lys His Ser Glu Gln Val Pro Asp Ile Leu 245 250 255 GlnLeu Asn Ala Ile Phe Asn Met Leu Asn Thr Lys Asn Cys Pro Ser 260 265 270Leu Lys Asp Lys Pro Lys Val Ile Ile Ile Gln Ala Cys Arg Gly Asp 275 280285 Ser Pro Gly Val Val Trp Phe Lys Asp Ser Val Gly Val Ser Gly Asn 290295 300 Leu Ser Leu Pro Thr Thr Glu Glu Phe Glu Asp Asp Ala Ile Lys Lys305 310 315 320 Ala His Ile Glu Lys Asp Phe Ile Ala Phe Cys Ser Ser ThrPro Asp 325 330 335 Asn Val Ser Trp Arg His Pro Thr Met Gly Ser Val PheIle Gly Arg 340 345 350 Leu Ile Glu His Met Gln Glu Tyr Ala Cys Ser CysAsp Val Glu Glu 355 360 365 Ile Phe Arg Lys Val Arg Phe Ser Phe Glu GlnPro Asp Gly Arg Ala 370 375 380 Gln Met Pro Thr Thr Glu Arg Val Thr LeuThr Arg Cys Phe Tyr Leu 385 390 395 400 Phe Pro Gly His 57 bases nucleicacid single linear oligonucleotide primer 3 GGGGAATTCA TGGCTATCAAAAAAGCTCAC ATCGAAAAAG ACTTCATCGC TTTCTGC 57 42 bases nucleic acid singlelinear oligonucleotide primer 4 TTCTGGCTGC TCAAATGAAA AACGAACCTTGCGGAAAATT TC 42 43 bases nucleic acid single linear oligonucleotideprimer 5 GGTTCGTTTT TCATTTGAGC AGCCAGAAGG TAGAGCGCAG ATG 43 35 basesnucleic acid single linear oligonucleotide primer 6 CCCCACTAGTCCTCTATTAA TGTCCTGGGA AGAGG 35

What is claimed is:
 1. A method for cleaving an interleukin-1βconverting enzyme (ICE) substrate, comprising contacting the substratewith a modified ICE such that the ICE substrate is cleaved, wherein themodified ICE comprises an amino acid sequence having an amino acidcorresponding to aspartic acid at position 381 of unmodified ICE (SEQ IDNO: 2) replaced with a mutant amino acid structure, the mutant aminoacid structure being capable of forming a salt bridge with an amino acidcorresponding to arginine at position 383 of unmodified ICE.
 2. Themethod of claim 1, wherein the mutant amino acid structure is glutamicacid.
 3. The method of claim 1, wherein the mutant amino acid structureis selected from the group consisting of serine, threonine, asparagineand glutamine.
 4. A method for identifying a modulator of interleukin-1βconverting enzyme (ICE) protease activity, comprising: preparing amodified ICE comprising an amino acid sequence wherein an amino acidcorresponding to aspartic acid at position 381 of unmodified ICE (SEQ IDNO: 2) is replaced with a mutant amino acid structure, the mutant aminoacid structure being capable of forming a salt bridge with an amino acidcorresponding to arginine at position 383 of unmodified ICE, such thatthe modified ICE retains proteolytic activity and exhibits increasedstability compared to unmodified ICE; contacting the modified ICE withan ICE substrate in the presence of a test compound under proteolyticconditions; measuring proteolysis of the ICE substrate in the presenceof the test compound; comparing proteolysis of the ICE substrate in thepresence of the test compound to proteolysis of the ICE substrate in theabsence of the test compound; and identifying the test compound as amodulator of ICE protease activity.
 5. The method of claim 4, whereinthe mutant amino acid structure is glutamic acid.
 6. The method of claim4, wherein the mutant amino acid structure is selected from the groupconsisting of serine, threonine, asparagine and glutamine.
 7. The methodof claim 4, wherein the test compound inhibits ICE protease activity. 8.The method of claim 4, wherein the test compound stimulates ICE proteaseactivity.
 9. A method for cleaving an interleukin-1β converting enzyme(ICE) substrate, comprising contacting the substrate with a modified ICEsuch that the ICE substrate is cleaved, wherein the modifiedinterleukin-1β converting enzyme (ICE) comprising an amino acid sequencewherein an amino acid corresponding to aspartic acid at position 381 ofunmodified ICE (SEQ ID NO: 2) is replaced with an amino acid selectedfrom the group consisting of serine, threonine, asparagine andglutamine.